Comparison of three magnetic resonance imaging measures of brain iron in healthy and cocaine use disorder participants.

Several magnetic resonance imaging (MRI) measures for quantifying endogenous nonheme brain iron have been proposed. These correspond to distinct physical properties with varying sensitivities and specificities to iron. Moreover, they may depend not only on tissue iron concentration, but also on the intravoxel spatial pattern of iron deposition, which is complex in many brain regions. Here, the three MRI brain iron measures of R 2 * , magnetic field correlation (MFC), and magnetic susceptibility are compared in several deep gray matter regions for both healthy participants (HPs) and individuals with cocaine use disorder (CUD). Their concordance is assessed from their correlations with each other and their relative dependencies on age. In addition, associations between the iron measures and microstructure in adjacent white matter regions are investigated by calculating their correlations with diffusion MRI measures from the internal capsule, and associations with cognition are determined by using results from a battery of standardized tests relevant to CUD. It is found that all three iron measures are strongly correlated with each other for the considered gray matter regions, but with correlation coefficients substantially less than one indicating important differences. The age dependencies of all three measures are qualitatively similar in most regions, except for the red nucleus, where the susceptibility has a significantly stronger correlation with age than R 2 * . Weak to moderate correlations are seen for the iron measures with several of the diffusion and cognitive measures, with the strongest correlations being obtained for R 2 * . The iron measures differ little between the HP and CUD groups, although susceptibility is significantly lower in the red nucleus for the CUD group. For the comparisons made, the iron measures behave similarly in most respects, but with notable quantitative differences. It is suggested that these differences may be, in part, attributable to a higher sensitivity to the spatial pattern of iron deposition for R 2 * and MFC than for susceptibility. This is supported most strongly by a sharp contrast between the values of the iron measures in the globus pallidus relative to those in the red nucleus. The observed correlations of the iron measures with diffusion and cognitive scores point to possible connections between gray matter iron, white matter microstructure, and cognition.

Atomistic simulations of the Escherichia coli ribosome provide selection criteria for translationally active substrates.

As genetic code expansion advances beyond L-α-amino acids to backbone modifications and new polymerization chemistries, delineating what substrates the ribosome can accommodate remains a challenge. The Escherichia coli ribosome tolerates non-L-α-amino acids in vitro, but few structural insights that explain how are available, and the boundary conditions for efficient bond formation are so far unknown. Here we determine a high-resolution cryogenic electron microscopy structure of the E. coli ribosome containing α-amino acid monomers and use metadynamics simulations to define energy surface minima and understand incorporation efficiencies. Reactive monomers across diverse structural classes favour a conformational space where the aminoacyl-tRNA nucleophile is <4 Å from the peptidyl-tRNA carbonyl with a Bürgi-Dunitz angle of 76-115°. Monomers with free energy minima that fall outside this conformational space do not react efficiently. This insight should accelerate the in vivo and in vitro ribosomal synthesis of sequence-defined, non-peptide heterooligomers.

Anionic G•U pairs in bacterial ribosomal rRNAs.

Wobble GU pairs (or G•U) occur frequently within double-stranded RNA helices interspersed between standard G=C and A-U Watson-Crick pairs. Another type of G•U pair interacting via their Watson-Crick edges has been observed in the A site of ribosome structures between a modified U34 in the tRNA anticodon triplet and G + 3 in the mRNA. In such pairs, the electronic structure of the U is changed with a negative charge on N3(U), resulting in two H-bonds between N1(G)…O4(U) and N2(G)…N3(U). Here, we report that such pairs occur in other highly conserved positions in ribosomal RNAs of bacteria in the absence of U modification. An anionic cis Watson-Crick G•G pair is also observed and well conserved in the small subunit. These pairs are observed in tightly folded regions.

mRNA decoding in human is kinetically and structurally distinct from bacteria.

In all species, ribosomes synthesize proteins by faithfully decoding messenger RNA (mRNA) nucleotide sequences using aminoacyl-tRNA substrates. Current knowledge of the decoding mechanism derives principally from studies on bacterial systems1. Although key features are conserved across evolution2, eukaryotes achieve higher-fidelity mRNA decoding than bacteria3. In human, changes in decoding fidelity are linked to ageing and disease and represent a potential point of therapeutic intervention in both viral and cancer treatment4-6. Here we combine single-molecule imaging and cryogenic electron microscopy methods to examine the molecular basis of human ribosome fidelity to reveal that the decoding mechanism is both kinetically and structurally distinct from that of bacteria. Although decoding is globally analogous in both species, the reaction coordinate of aminoacyl-tRNA movement is altered on the human ribosome and the process is an order of magnitude slower. These distinctions arise from eukaryote-specific structural elements in the human ribosome and in the elongation factor eukaryotic elongation factor 1A (eEF1A) that together coordinate faithful tRNA incorporation at each mRNA codon. The distinct nature and timing of conformational changes within the ribosome and eEF1A rationalize how increased decoding fidelity is achieved and potentially regulated in eukaryotic species.

Rare ribosomal RNA sequences from archaea stabilize the bacterial ribosome.

The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3'-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

Structure of the bacterial ribosome at 2 Å resolution.

Using cryo-electron microscopy (cryo-EM), we determined the structure of the Escherichia coli 70S ribosome with a global resolution of 2.0 Å. The maps reveal unambiguous positioning of protein and RNA residues, their detailed chemical interactions, and chemical modifications. Notable features include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former likely conserved in all domains of life. The maps also reveal extensive solvation of the small (30S) ribosomal subunit, and interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The maps and models of the bacterial ribosome presented here now allow a deeper phylogenetic analysis of ribosomal components including structural conservation to the level of solvation. The high quality of the maps should enable future structural analyses of the chemical basis for translation and aid the development of robust tools for cryo-EM structure modeling and refinement.

Inside cells, proteins are produced by complex molecular machines called ribosomes. Techniques that allow scientists to visualize ribosomes at the atomic level, such as cryogenic electron microscopy (cryo-EM), help shed light on the structure of these molecular machines, revealing details of how they build proteins. Understanding how ribosomes work has many benefits, including the development of new antibiotics that can kill bacteria without affecting animal cells. Watson et al. used cryo-EM techniques with increased resolution to examine the ribosomes of the bacterium Escherichia coli in a higher level of detail than has been seen before. The results revealed two chemical modifications in proteins that form the ribosome that had not been observed in ribosomes previously. Additionally, a protein segment with a previously undescribed structure was identified close to the site where the ribosome reads the genetic instructions needed to make proteins. Further genetic analyses suggested these structures are in many related species, and may play important roles in how the ribosome works. Watson et al. were also able to see how paromomycin, an antibiotic used to treat parasitic infections, is positioned in the ribosome. The antibiotic interacts with a site near where the genetic code is read out, which might explain why certain changes to the antibiotic can interfere with its potency. Finally, the new ribosome structure reveals thousands of water molecules and metal ions that help keep the ribosome together as it produces proteins. This study shows the value of advances in cryo-EM technology and illustrates the importance of applying these techniques to other cell components. The results also reveal details of the ribosome useful for further research into this essential molecular machine.

Sleep, physical activity, waist circumference and diet as factors that influence health for reproductive age women in northern Greenland.

BACKGROUND: This study explored community and individual factors that influence the health of reproductive age women in a settlement in northern Greenland. This is important because Greenland has a declining population, a high abortion rate and because of projected environmental shifts due to climate change. METHODS: This study collected mixed methods data to explore diet, physical activity, sleep and waist circumference for reproductive age women in Kullorsuaq, Greenland. The daily steps and sleeping hours of 13 reproductive age women were measured using activity monitoring bracelets. Waist circumference measurements and in-depth interviews about daily eating and physical activity were conducted with 15 participants and ethnographic participant observations were recorded using field notes. RESULTS: Waist circumference measurements were above recommended cutoffs established by the World Health Organization. Physical activity measured by daily steps was within the 'active' range using the cutoff points established by Tudor and Locke. Physical activity is social and is important for communal relationships. Sleeping hours were within normal ranges based on US guidelines; however, the quality of this sleep, its variability across seasons and cultural expectations of what healthy sleep means must be further explored. Diets of women included a mixture of locally harvested meats and imported packaged foods. Study participants experienced less satiety and reported getting hungrier faster when eating packaged foods. This research took place in Spring 2016 and women reported that their sleep, physical activity and diet fluctuate seasonally. CONCLUSION: The reported findings suggest further investigation of sleep, diet and physical activity combined with the measurement of reproductive hormones to determine linkages between lifestyle factors and reproductive health outcomes is needed.

Defects in the Assembly of Ribosomes Selected for ß-Amino Acid Incorporation.

Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve the incorporation of several nonstandard monomers including d-amino acids, dipeptides, and ß-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these ribosomes catalyze incorporation of these new substrates. Here, we probed the properties of a mutant ribosome-P7A7-evolved for better in vivo ß-amino acid incorporation through in vitro biochemistry and cryo-electron microscopy. Although P7A7 is a functional ribosome in vivo, it is inactive in vitro, and assembles poorly into 70S ribosome complexes. Structural characterization revealed large regions of disorder in the peptidyltransferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient ribosome assembly during ribosome engineering toward new catalytic abilities.

Structure of ribosome-bound azole-modified peptide phazolicin rationalizes its species-specific mode of bacterial translation inhibition.

Ribosome-synthesized post-translationally modified peptides (RiPPs) represent a rapidly expanding class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants. The cryo-EM structure of the Escherichia coli 70S-PHZ complex reveals that the drug interacts with the 23S rRNA and uL4/uL22 proteins and obstructs ribosomal exit tunnel in a way that is distinct from other compounds. We show that the uL4 loop sequence determines the species-specificity of antibiotic action. PHZ expands the known diversity of LAPs and may be used in the future as biocontrol agent for agricultural needs.

The Grocery Store Food Environment in Northern Greenland and Its Implications for the Health of Reproductive Age Women.

The population of Greenland is diminishing and environmental and social shifts implicate food availability and the health of reproductive age women. There is little knowledge of the grocery store food environment in Greenland. To address this gap and provide baseline information the present study measured food availability in five grocery stores in northern Greenland. As well, 15 interviews were conducted with reproductive age women, three grocery store managers were interviewed and one interview was conducted with a food distribution manager. Results show few fresh fruits and vegetables are available in grocery stores and in some stores no fresh foods are available. In Kullorsuaq, the primary location for this study, the Nutrition Environment Measures Survey in Stores score in spring 2016 was (3/30) and the Freedman Grocery Store Survey Score was (11/49). Interview results highlight a need to increase communication within the food system and to tailor food distribution policies to the Arctic context with longer term planning protocols for food distribution. These findings can be used to inform future food store environment research in Greenland and for informing policies that improve healthful food availability in grocery stores in northern Greenland.

Monolayer-crystal streptavidin support films provide an internal standard of cryo-EM image quality.

Analysis of images of biotinylated Escherichia coli 70S ribosome particles, bound to streptavidin affinity grids, demonstrates that the image-quality of particles can be predicted by the image-quality of the monolayer crystalline support film. The quality of the Thon rings is also a good predictor of the image-quality of particles, but only when images of the streptavidin crystals extend to relatively high resolution. When the estimated resolution of streptavidin was 5Å or worse, for example, the ribosomal density map obtained from 22,697 particles went to only 9.5Å, while the resolution of the map reached 4.0Å for the same number of particles, when the estimated resolution of streptavidin crystal was 4Å or better. It thus is easy to tell which images in a data set ought to be retained for further work, based on the highest resolution seen for Bragg peaks in the computed Fourier transforms of the streptavidin component. The refined density map obtained from 57,826 particles obtained in this way extended to 3.6Å, a marked improvement over the value of 3.9Å obtained previously from a subset of 52,433 particles obtained from the same initial data set of 101,213 particles after 3-D classification. These results are consistent with the hypothesis that interaction with the air-water interface can damage particles when the sample becomes too thin. Streptavidin monolayer crystals appear to provide a good indication of when that is the case.

Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules.

We describe a rapid and convenient method of growing streptavidin (SA) monolayer crystals directly on holey-carbon EM grids. As expected, these SA monolayer crystals retain their biotin-binding function and crystalline order through a cycle of embedding in trehalose and, later, its removal. This fact allows one to prepare, and store for later use, EM grids on which SA monolayer crystals serve as an affinity substrate for preparing specimens of biological macromolecules. In addition, we report that coating the lipid-tail side of trehalose-embedded monolayer crystals with evaporated carbon appears to improve the consistency with which well-ordered, single crystals are observed to span over entire, 2µm holes of the support films. Randomly biotinylated 70S ribosomes are used as a test specimen to show that these support films can be used to obtain a high-resolution cryo-EM structure.

Electronic and optical properties of polypyridylruthenium derivatized polystyrenes: multi-level computational analysis of metallo-polymeric chromophore assemblies.

Great effort is geared toward investigation of new materials for solar energy conversion in recent years. Polymeric chromophore assemblies consisting of [Ru(bpy)3](2+) complexes attached to a polystyrene backbone have gained considerable interest in recent years because of their structural flexibility combined with their ability to efficiently capture solar energy and transport the captured energy in the form of exciton or charges. We employ a combination of computational methods to examine how opto-electronic properties of [Ru(bpy)3](2+) complexes are influenced by the polymer dynamics in these polymeric chromophore assemblies. The covalent linker between the polymer and the light-absorbing Ru complex is thought to play an important role in optimizing the assemblies for solar energy conversion and transport. We find that the presence of -CH2- groups in the linker has a significant impact on the Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO) energies of the pendants. Generally speaking, a longer linker leads to higher HOMO energies. Without the presence of -CH2- groups, a mixture of cis and trans amide bond in the covalent linker leads to a bimodal distribution for both HOMO and LUMO energies. Importantly, we find that distributions of orbital energies from individual [Ru(bpy)3](2+) pendants have the maximum overlap when there is only one -CH2- group in the linker. Such an isotropic energy distribution is likely to be important for charge transport within the assemblies. We also find that in contrast to the isolated [Ru(bpy)3](2+) complex, the HOMO is generally found on the linker rather than on Ru atom. This does not change the character of the metal-to-ligand charge transfer (MLCT) excited states, as these excitations in the pendants do not derive from HOMO/LUMO transitions but rather from HOMO - 2/LUMO transition since HOMO - 2 is located on the Ru atom.

Atom transfer radical polymerization preparation and photophysical properties of polypyridylruthenium derivatized polystyrenes.

A ruthenium containing polymer featuring a short carbonyl-amino-methylene linker has been prepared by atom transfer radical polymerization (ATRP). The polymer was derived from ATRP of the N-hydroxysuccinimide (NHS) derivative of p-vinylbenzoic acid, followed by an amide coupling reaction of the NHS-polystyrene with Ru(II) complexes derivatized with aminomethyl groups (i.e., [Ru(bpy)2(CH3-bpy-CH2NH2)](2+) where bpy is 2,2'-bipyridine, and CH3-bpy-CH2NH2 is 4-methyl-4'-aminomethyl-2,2'-bipyridine). The Ru-functionalized polymer structure was confirmed by using nuclear magnetic resonance and infrared spectroscopy, and the results suggest that a high loading ratio of polypyridylruthenium chromophores on the polystyrene backbone was achieved. The photophysical properties of the polymer were characterized in solution and in rigid ethylene glycol glasses. In solution, emission quantum yield and lifetime studies reveal that the polymer's metal-to-ligand charge transfer (MLCT) excited states are quenched relative to a model Ru complex chromophore. In rigid media, the MLCT-ground state band gap and lifetime are both increased relative to solution with time-resolved emission measurements revealing fast energy transfer hopping within the polymer. Molecular dynamics studies of the polymer synthesized here as well as similar model systems with various spatial arrangements of the pendant Ru complex chromophores suggest that the carbonyl-amino-methylene linker probed in our target polymer provides shorter Ru-Ru nearest-neighbor distances leading to an increased Ru*-Ru energy hopping rate, compared to those with longer linkers in counterpart polymers.